Fluorescent Dyes and Complexes

ABSTRACT

A fluorescent dye comprising a xanthene-derived fluorophore having the formula (I) wherein R 1 , R 2 , R 3 , R 4 , R 5  and R 6  are independently selected from H, alkyl, alkoxy, alcohol, ether, alkenyl, alkenoxy, aryl, alkaryl, aralkyl and amido, except that R 1 , R 4  and/or R 5  is not H when bonded to Y, Y 1  or Y 2 , respectively; X is either O −  or S − ; and at least one of Y, Y 1 , Y 2 , Y 3 , Y 4  and Y 5  is a group for covalently bonding the dye, optionally through. the use of a coupling agent, to a target molecule, and is otherwise H. The dye may be covalently attached to a target molecule to form a complex, and the dye and/or complex finds use in cell analysis techniques, particularly pH measurement and analysis of kinetics of migration.

FIELD OF THE INVENTION

This invention relates to fluorescent dyes comprising a xanthene derivedfluorophore, methods of making the same, complexes comprising thesedyes, and use of such dyes and complexes in cell analysis procedures.

BACKGROUND OF THE INVENTION

Fluorescent dyes, when bonded to target molecules such as proteins, arecommonly used to probe the properties of living cells. In particular,such dyes are often used to probe the pH of individual cellcompartments. This is commonly achieved by comparing the intensity offluorescence of a particular dye when it is within a particular cellcompartment with a calibration curve of intensity versus pH for thatdye.

In some instances, for example when greater accuracy is required, twodyes (with greatly different responses at the measured pH) are used. Theratio of the two observed intensities is compared to a calibrationcurve, to determine a pH value more reliably.

It has now been realised that even greater accuracy can be achieved bytailoring the most sensitive region of a dye's pH response to theexpected pH of the cell or cell compartment. This can be done by using adye whose PK_(a) is approximately equal to the expected pH of the cellor cell compartment.

At present, long wavelength, fluorescent dyes bonded to suitable targetproteins have pK_(a)'s of about 6 and above. However, some cellcompartments of interest have a pH of less than 6. It would therefore bedesirable to provide dyes which have pK_(a)'s of less than 6 when boundto protein, thus allowing their pK_(a) to be matched to the approximatepH of their target cell compartment.

SUMMARY OF THE INVENTION

According to a first embodiment of the present invention, a fluorescentdye comprises a xanthene-derived fluorophore having the formula (I)

wherein

-   -   R¹, R², R³, R⁴, R⁵ and R⁶ are independently selected from H,        alkyl, alkoxy, alcohol, ether, alkenyl, alkenoxy, aryl, alkaryl,        aralkyl and amido, except that R¹, R⁴ and/or R⁵ is not H when        bonded to Y, Y¹ and/or Y², respectively;    -   X is either O⁻ or S⁻; and

at least one of Y, Y¹, Y², Y³, Y⁴ and Y⁵ is a group for covalentlybonding the dye, optionally through the use of a coupling agent, to atarget molecule, and is otherwise H.

According to a second embodiment of the present invention, a method formaking a dye of the type defined above comprises reacting ameta-aminophenol with a carboxylate compound capable of undergoingalpha-cleavage, e.g. a beta-ketocarboxylate.

According to a third embodiment of the present invention, a method formaking a dye having the formula (I) defined above comprises reacting acompound having the formula (II)

with a compound of formula (CO)R⁷ ₂ wherein R⁷ is a leaving group, toform an intermediate having the formula (III)

wherein R¹, R², R³, R⁴ and Y are as defined for formula (I), andconverting the intermediate to a dye having the formula (I).

According to a fourth embodiment of the present invention, a method formaking a dye having the formula (I) as defined above comprises reactinga compound having the formula (II), above, with a benzaldehyde, benzoicacid, activated benzoic acid or benzoate, wherein when reaction is witha benzaldehyde the method further comprises post-condensation oxidationto form a dye having the formula (I).

According to a fifth embodiment of the present invention, a method formaking a dye having the formula (I) as defined above comprises reactingtogether a para-amino substituted salicylaldehyde and a meta-aminophenolto form a compound having the formula (IIa), wherein R⁸ is H or an alkylgroup; cyclising the compound of formula (IIa) to form a compound havingthe formula (IIb); reacting the compound having the formula (IIb) with ameta-aminophenol to form a dye having the formula (IIc); and oxidisingthe compound having the formula (IIc) to form a dye having the formula(I).

According to a sixth embodiment of the present invention, a method formaking a dye having the formula (I) as defined above comprises reactingtogether a para-amino substituted salicylaldehyde and a meta-aminophenolin aqueous or alcoholic solution and in the presence of acid, to form acompound having the formula (IIa), above; cyclising the compound havingthe formula (IIa) to form a compound having the formula (IIb), above;converting the compound having the formula (IIb) to a compound havingthe formula (III), above; and converting the compound having the formula(III) to a dye having the formula (I).

According to a seventh embodiment of the present invention, a method formaking a dye having the formula (I) as defined above comprises reactingtogether a meta-aminophenol with a compound having the formula

wherein X is a halogen, a B(OR⁹)₂ group in which R⁹ is H or an alkylgroup, or another leaving group, to form a compound having the formula(II), above, and converting the compound having the formula (II) to adye having the formula (I) by:

a) reaction with a salicylic ester;

b) reaction with a salicylaldehyde, and subsequent oxidation; or

c) converting to a compound having the formula (II) above, and thenconverting this to a dye having the formula (I).

According to an eighth embodiment of the present invention, afluorescent complex comprises a dye having the formula (I) as definedabove, which is bonded to a target molecule through a group selectedfrom Y, Y¹, Y², Y³, Y⁴ and Y⁵, which is not H, optionally through theuse of a coupling agent.

The dyes and complexes of the present invention show a negativefluorescence intensity response with increasing pH. This means that theintensity of fluorescence exhibited by the dyes and complexes decreaseswith increasing (i.e. more alkaline) pH. Furthermore, the pK_(a)'s ofthese dyes and complexes may be varied by selection of the substituentsR⁵ and R⁶, and by the inclusion of electron donating groups on thelowermost aryl ring, to give a range of dyes and complexes havingpK_(a)'s particularly suited to cell analysis procedures.

According to a ninth embodiment of the present invention, a living cellor cell compartment comprises a complex as defined above, either bondedthereto or contained therein.

According to further embodiments of the present invention, such dyes andcomplexes are used, either alone or in combination with other dyes orcomplexes, to establish the pH of a living cell or cell compartment, orto analyse the kinetics of migration of the dyes or complexes into aliving cell or cell compartment, or from location to location within acell. Yet another embodiment of the invention comprises a novel methodof making intermediates en route to the dyes of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Dyes according to the present invention are based on a rhodamine-typestructure, and as such comprise a xanthene-derived fluorophore and anadditional aryl ring. The xanthene-derived fluorophore and the aryl ringare mutually perpendicular, and therefore no conjugation exists betweenthem.

In formula (I), the groups R¹, R², R³ and R⁴ on the xanthene moiety areindependently selected from H (except that R¹ and/or R⁴ can not be H ifY or Y¹ is bonded thereto, respectively), alkyl, alkoxy, alcohol, ether,alkenyl, alkenoxy, aryl, alkaryl, aralkyl and amido. The size of thesegroups is limited only by steric considerations, and the need forcompatibility with whichever method of coupling the dye to a targetmolecule is desired. Typically, however, these groups comprise up to 20carbon atoms. Preferably, these groups are independently selected fromalkyl groups, more preferably alkyl groups having up to 10 carbon atoms.

For clarity, in the context of the present Application, reference togroups such as alkyl, alkoxy etc. include substituted and unsubstitutedgroups. Furthermore, any of the aryl rings may include additionalsubstituents not shown in formula (I).

Where water solubility of the dye is required, for instance to allowbonding to a protein without denaturation of the protein, one or more ofR¹, R², R³ and R⁴ are selected from or include polar groups such asalcohol, alkoxy, ether, amido, carboxylate, sulphonate, amino, ammoniumetc.

The aryl ring of the dye bears two substituents, X and NR⁵R⁶. The Xgroup is ortho to the xanthene moiety, and is believed responsible forthe pH response of the dye. X may be selected from O⁻ and S⁻, but ispreferably O⁻. The protonated forms of the group X, ie. OH and SH, arealso encompassed by the present invention.

The NR⁵R⁶ substituent can take any available position on the phenylring, although preferably it is para to the xanthene moiety. R⁵ and R⁶are independently selected from H (unless Y¹ is bonded to R⁵, alkyl,alkoxy, alcohol, ether, alkenyl, alkenoxy, aryl, alkaryl or aralkyl oramido. More preferably, however, R⁵ and R⁶ are independently selectedfrom alkyl, alcohol or ether groups, and are most preferably alkylgroups. As for groups R¹ to R⁴, the size of groups R⁵ and R⁶ is limitedonly by steric considerations, and the need for compatibility withwhichever method of coupling the dye to a target molecule is desired.Typically, however, these groups comprise up to 20 carbon atoms, andmore typically up to 10 carbon atoms.

A particularly preferred dye comprises X as O⁻ (or OH) and each of R¹,R², R³, R⁴, R⁵ and R⁶ independently selected from alkyl groups.

Optionally, the aryl ring may comprise one or more additionalsubstituents which may occupy any available position on the ring. It ispreferred that such substituents are electron donating in nature, unlessthe substituent is for bonding to a target molecule, as is describedbelow. Alkyl groups, typically having from 1 to 10 carbon atoms, arepreferred electron donating substituents.

The dyes are functionalised so that they may be bonded to a range oftarget molecules, for introduction into a living cell or cellcompartment. If desired, a plurality of different dyes, havingfluorescence responses at different pH's, may be bonded to a singletarget molecule. This may be particularly useful for analysing thekinetics of migration of a particular target molecule through differentregions of a cell, or through different cell compartments.

In the context of the present Application, a living cell includeseukaryotic cells, prokaryotic cells and plant cells. Further, while thepresent invention is described in the context of use of the novelfluorescent dyes with living cells, the present invention may find usein other applications, and in particular in the analysis ofnon-biological systems.

Suitable target molecules include peptides, polypeptides, proteins,sugars (i.e. saccharides and polysaccharides) and antibodies.

There is a wide variety of functionalities that may be included in thedyes of the present invention to achieve bonding to target molecules.For clarity, bonding may be achieved by direct reaction between a dyeand its target molecule, herein referred to as “direct bonding”.Alternatively, “indirect bonding” may be achieved with the aid of acoupling agent, or “activator”, which activates the dye by forming acomplex therewith, and which is typically displaced after reaction withthe target molecule. Depending on the nature of the coupling agent,however, a moiety derived therefrom may become incorporated in the finalbonded complex. A wide variety of coupling reactions is known in theprior art, and may be applicable to the present invention.

Bonding to a target molecule may take place through any one of groups Y,Y¹, Y², Y³, Y⁴ or Y⁵, when these are not H, and which hereinafter arereferred to collectively as the “bonding groups”. A single dye maycontain more than one bonding group suitable for bonding to a targetmolecule, but generally bonding only occurs through one of these groups.

Generally, it is preferred that the dye be bonded to a target moleculethrough a substituent on the xanthene moiety, i.e. Y, Y¹, Y³ or Y⁴,rather than through Y² or Y⁵ as the substituent —NR⁵R⁶ is believed toaffect the pK_(a) of the dye or complex, as will be described in moredetail below, and other substituents on the same aryl ring may have asimilar effect.

Generally, if bonding to a target molecule is to be achieved througheither of groups Y³ or Y⁴, steric considerations need to be taken intoaccount. Typically group Y³ and/or Y⁴ will be ortho or meta, preferablyortho, to the nitrogen-containing group, NR¹R² or NR³R⁴. If bonding isto be achieved through a group located meta to this group it ispreferred that bonding should not be via a hetero atom, as this mayreduce or remove the fluorescence response.

The nature of the bonding group, or groups, will depend upon the natureof the functional groups on the target molecule available for reactionwith the dye. For instance, where the target molecule contains pendantamino or amine groups, the bonding group may be selected fromisocyanates; isothiocyanates; and carbonyl groups containing a leavinggroup, for instance acid chloride, sulphonate, carboxylate, andso-called “active” esters, i.e. containing very good leaving groupsincluding, for instance, nitrophenyl and N-hydroxysuccinimide, all ofwhich are capable of direct bonding to said amino or amine groups on thetarget molecule. Alternatively, the bonding group may comprise acarboxylic acid group, an aldehyde or a ketone which, on activation witha coupling agent, are capable of reaction with said amino or aminegroups. When the bonding group is an aldehyde or ketone, the reaction istypically conducted under reducing conditions to give rise to anamino-alkyl chain linkage in the final dye/target molecule complex.

Where the target molecule comprises a thiol group available for reactionwith the dye, the bonding group may be selected from many of the groupsmentioned above, disulphide and thiols.

For clarity, many of the above bonding groups will react with and bondto a wide variety of target functional groups on target molecules, i.e.other than amino, amine and thiol groups, optionally through the use ofa coupling agent.

In case the bonding group is an additional substituent on one of thearyl rings of the xanthene moiety, preferred bonding groups includecarboxylic acids, ethers and groups attached to the ring(s) through analkyl group.

For cell analysis procedures, it is preferred that the dyes or complexesof the present invention have a range of pK_(a)'s in the range 1 to 7,more preferably 2 to 5.5. As mentioned above, the pK_(a)'s of these dyesappear to be primarily governed by the R⁵ and R⁶ substituents, and anyadditional substituents, on the aryl ring. For example, it seems thatthe pK_(a)'s of these dyes increase as R⁵ and R⁶ are progressed throughprimary to secondary to tertiary alkyl substituents. This finding issurprising, since, as discussed above, the aryl ring is not conjugatedwith the xanthene moiety. This lack of conjugation would be expected tominimise any effect of the R⁵, R⁶ and other substituents on the energylevels of the xanthene fluorophore, and hence minimise any changes tothe fluorescence response of this fluorophore, consequently minimisingany change in pK_(a).

By way of illustration, Table 1, below, lists a number of dyes, someprior to functionalisation, and complexes according to the presentinvention (in each X is O⁻), together with their pK_(a) values. In Table1, and throughout the present Application, Me denotes a methyl group, Etan ethyl group and Ph a phenyl group.

The dyes according to the present invention can be prepared by anycombination of standard chemical synthetic steps. In the following, thesyntheses described focus on the preparation of dyes in which the NR⁵R⁶substituent is para to the xanthene moiety, but may be modified ifnecessary by routine measures to prepare dyes having this substituent inother positions.

One method of preparation of the dyes of the present invention involvesreaction of a meta-aminophenol (or a mixture of meta-aminophenols) witha carboxylate compound capable of undergoing alpha-cleavage, forinstance a beta-ketocarboxylate. A preferred beta-ketocarboxylate isdiethylmalonate. Another example of a carboxylate compound capable ofundergoing alpha-cleavage suitable for use in the present invention is5-nitrosalicylic acid methyl ester.

The reaction proceeds via an intermediate having the formula (III),below, wherein R¹, R², R³, R⁴ and Y are as defined for formula (I),which on further reaction with the carboxylate compound is converted toa dye having the formula (I).

Another method of preparing the dyes according to the present inventioncomprises reacting a compound having the formula (II) above, with acompound of formula COR⁷ ₂, wherein R⁷ is a leaving group, to form anintermediate having the formula (III) above, and converting theintermediate to the desired dye by reaction with a meta-aminophenol (ora mixture of meta-aminophenols), or a derivative thereof, or by standardchemical steps. One such combination of standard chemical stepscomprises reducing the carbonyl group of the intermediate and turningthe resulting OH group into a leaving group; condensing with a suitablemolecule; and then reoxidising to a dye having the formula (I).

Yet another method for preparing the dyes of the present inventioncomprises reacting a compound having the formula (II), above, with abenzaldehyde, benzoic acid, activated benzoic acid, or benzoate. Wherethe method comprises reaction with a benzaldehyde the resulting productmust be oxidised to produce a dye having the formula (I). In the contextof the present Application, the term “activated benzoic acid” isintended to cover benzoic acid derivatives such as benzoic acidchlorides.

Yet another method for preparing the dyes of the present invention,which is particularly useful for preparing dyes in which the aryl ringsof the xanthene moiety are different to one another, comprises reactingtogether a para-amino substituted salicylaldehyde and a meta-aminophenolto form a compound having the formula (IIa), as shown above. Thereaction is typically conducted in aqueous or alcoholic solution and inthe presence of acid. Dilute acid may be used, although stronger acidsmay avoid the need for heat to drive the reaction. Examples of suitableacids include hydrochloric acid and phosphoric acid. If the reactiontakes place in an alcoholic solution this may result in R⁸ being otherthan H, and typically it will be an alkyl group derived from the alcoholpresent. Any liquid alcohol may be used, with the lower (C₁₋₆) alcohols,such as methanol, ethanol, propanol and butanol, being preferred.

Alternatively, the salicylaldehyde might be replaced by a para-aminosubstituted ester of salicyclic acid.

The next step in this method requires cyclisation of the compound havingthe formula (IIa) to a compound having the formula (IIb), again as shownabove. Cyclisation is typically acid catalysed. The compound having theformula (IIb) may then be reacted in a number of ways, either a) to forma compound having the formula (IIc), again as above, followed byoxidation to the desired dye of formula (I), or b) oxidation to acompound having the formula (III), with subsequent conversion to thedesired dye of formula (I). If, as is preferred, the reaction is toproceed via a compound having the formula (IIc), this requires reactionwith further meta-aminophenol, selected according to the desiredproduct. If the reaction is to proceed via a ketone of formula (III),this may be obtained from compound (IIb) using the methodology describedby Ehrlich et al, Chem. Ber (1913)46:1941. Essentially, this involvessubstituting the group —OR⁸ with chlorine, reacting with a cyamide saltto replace chlorine with a nitrile group, and oxidising to form aketone.

Yet another method for preparing the dyes of the present invention,which is again suitable for preparing dyes in which the aryl groups ofthe xanthene moiety differ from one another, comprises reacting togethera meta-aminophenol with a compound having the formula

wherein X is a halogen, such as Cl, Br or I, a B(OR⁹)₂ group in which R⁹is H or an alkyl group, or another leaving group. The reaction istypically conducted in the presence of a base and a copper compound.Suitable copper compounds include the cuprous salts CuI, Cu₂O and CuOAc(where Ac is acetate), and copper bronze. Platinum salts are alsosuitable for use in this reaction. Suitable bases include inorganicbases such as sodium or potassium carbonate, and organic bases such asdi-isopropylethylamine (DIPE).

The resulting product, having the formula (II) above, may be convertedto the desired dye by a) reaction with a salicylic ester; b) reactionwith a salicylaldehyde having the formula (IIc), above, and subsequentoxidation; or c) converting to a compound having the formula (III),above, and then to the desired dye.

Choice of reaction conditions and reagents for use in all these methodsare well within the capability of the skilled practitioner.

The dyes and complexes of the present invention can be used to determinethe pH of living cells or cell compartments. By “a cell compartment”typically we mean one of the many organelles suspended in the cellcytoplasm. The pH of a cell or cell compartment can be measured byintroducing a dye or complex into a cell or cell compartment,irradiating the dye or complex with a suitable light source, andobserving the intensity of fluorescence of the dye or complex. Theobserved fluorescence intensity can then be used to determine pH by avariety of methods known in the field, selected according to the methodof accumulation of the dye or complex. For instance, the observedfluorescence may be compared to a known standard, for example acalibration curve of fluorescence intensity versus pH, or tofluorescence intensity measurements indicative of the total dye orcomplex present. Any conventional fluorimetric equipment can be used toirradiate the sample, and to measure the resulting fluorescent response.

Typically, the dyes and complexes are introduced into a living cell orcell compartment by mixing with a sample comprising a cell or cellcompartment, and then leaving the mixture to stand for a time intervaladequate to allow entry of the dye or complex into the cell or cellcompartment. During this time interval, the dye or complex diffusestowards a cell or cell compartment within the sample. The dye or complexthen attaches itself to the membrane of a cell or cell compartment.

In the case of complexes, target molecules are generally cell or cellcompartment specific, hence a specific complex generally attaches toonly one kind of cell or cell compartment. Once attached to a cell orcell compartment, the dye or complex may diffuse through a membrane ofthat cell or cell compartment or be trafficked to a specific cellcompartment by receptor-mediated endocytosis, hence exposing itself tothe internal pH of the cell or cell compartment.

The dyes and complexes of the present invention allow more accuratedetermination of pH as compared to existing dyes because the pK_(a)'s ofthe dyes and complexes of the present invention can, by design, beadjusted by substitution to a variety of pK_(a) values. Thus, some aretuned to the pH of the cell or cell compartment of interest, andconsequently will be ideal for measuring the pH of a cell or cellcompartment when accumulated by receptor-mediated endocytosis or anynon-passive accumulation mechanism. Others will have a pKa far from thepH of the cell or cell compartment of interest, and will be ideal formeasuring pH when accumulation occurs by passive accumulation. Thissituation is best understood by considering these two mechanisms ofaccumulation of the dye or complex into the cell or cell compartment.

First, if the dye or complex is accumulated only passively according tothe pH difference between the pH of the cell or cell compartment to beinvestigated and the pH outside the cell or cell compartment,respectively, then the accuracy of pH measurement with a dye or complexis highest when the pK_(a) of the dye or complex is far from the pH tobe measured. In this situation one is essentially measuring theaccumulation at equilibrium as reported by fluorescence. Accumulationwill occur passively when one form of the dye or complex with respect topH (the uncharged form) freely penetrates the cell or cell compartmentof interest and the other form (a charged form) is non-penetrating.Fluorescence will approach its equilibrium position provided the form ofthe dye accumulated is the fluorescent form and that accumulation toequilibrium has occurred. The observed fluorescence intensity can thenbe used to determine pH according to any of the known methods, forinstance by reference to calibration data, or by comparing the observedfluorescence intensity to the fluorescence intensity observed onacidifying the test sample so that all the dye or complex fluoresces,the ratio of the two fluorescence intensities coupled with the known pKaallowing determination of pH. Passive accumulation can be achieved byuse of a dye that is not attached to a target molecule or a dye that isattached to a small, relatively hydrophobic target molecule capable ofdiffusing through the cell membrane.

Second, whenever the dye or complex is accumulated in the cell or cellcompartment by a mechanism that does not rely solely on passiveaccumulation, the accuracy of a pH measurement will be highest when thepK_(a) of the dye is close to the pH to be measured. The increasedaccuracy available with the dyes and complexes of the invention in thissituation arises from the fact that the pK_(a) is the pH of the aqueousmedium containing a species when it is 50% protonated and that at thispH a change in proton intensity will have greatest effect on theproperties of the species. Hence, the greatest change in fluorescenceintensity occurs at the pK_(a) of the dye, and measurements of absolutefluorescence intensity at this pH will give rise to more accurate pHreadings. That said, provided the titration range of the dye or complexused to analyse a particular cell or cell compartment embraces the pH ofthat cell or cell compartment, that is generally sufficient.

In the context of the present Application, a pKa is “far” from the pH tobe measured when different by more than 1 pH unit, and preferably morethan 2 pH units. For instance, if the dye or complex is a weak acid itspKa should be more than 1 pH unit below the pH to be measured, and ifthe dye or complex is a weak base then its pKa should be more than 1 pHunit above the pH to be measured. Further, in the context of the presentApplication, a pKa is “close” to the pH to be measured when they arewithin about 1 pH unit of each other.

Accuracy can be further increased by using a plurality of fluorescentdyes or complexes having different fluorescent responses. For instance,two or more dyes according to the invention may be used, optionallybonded to identical target molecules, or a dye according to theinvention and another dye. In one embodiment, the second fluorescent dyeor complex has a positive fluorescence response with increasing pH (bythis we mean that the intensity of fluorescence exhibited by the dye orcomplex increases with increasing pH). It is preferable that the two ormore dyes or complexes have overlapping titration ranges, and morepreferably the different dyes or complexes have pK_(a) values withinabout 1 unit of each other. The intensity of fluorescence of each dye orcomplex is then measured, and pH determined by calculating the ratio:

Fluorescence Intensity of the First Dye or Complex

Fluorescence Intensity of the Second Dye or Complex

and comparing the value obtained to a calibration curve.

According to another embodiment of the present invention, the dyes orcomplexes of the invention can be used to analyse the kinetics ofmigration of a species into or through a cell or cell compartment. Thisis done by monitoring the intensity of fluorescence of a dye or complexover a time interval. Where pH is known, the dye or complex should beselected so as to have a pK_(a) in the range between the pH at thestarting point and the pH at the end point of the pathway to beanalysed. In some cases it may be desirable to use a plurality of dyesor complexes having a variety of pK_(a)'s, with each dye or complextuned to a different portion of the pathway to be analysed.

According to yet another embodiment of the present invention, novel dyesare defined by formula (IV), below, wherein R¹, R², R³, R⁴, R⁵ and R⁶are independently selected from H, alkyl, alkoxy, alcohol, ether,alkenyl, alkenoxy, aryl, alkaryl, aralkyl and amido; and X is either O⁻or S⁻; with the proviso that when X is O⁻, and preferably irrespectiveof the nature of X, not all of R¹, R², R³, R⁴, R⁵ and R⁶ are the samealkyl group, unless an electron withdrawing group is present in the arylring having the —NR⁵R⁶ group, eg. para to the group X.

According to yet another embodiment, when X is O⁻ in formula (IV), notall of R¹, R², R³, R⁴, R⁵ and R⁶ are alkyl.

The preferred definitions of the various substituents are as for thedyes of formula (I) above, and additional substituents may be includedon any of the aryl rings. A preferred substituent is para to the group X(which is preferably O⁻) and is selected from electron donating groups,for instance alkyl groups, typically C₁₋₆ alkyl groups.

The dyes of formula (IV) may be prepared by any of the methods describedabove for preparation of the dyes having the formula (I). Furthermore,these methods my be used for the preparation of dyes having the formula(IV) even when X is O⁻ and each of R¹, R², R³, R⁴, R⁵ and R⁶ are thesame or different alkyl groups.

The dyes of formula (IV) may be functionalised by standard chemicalsteps to give dyes having the formula (I) above, and may then be bondedto target molecules for cell analysis procedures. TABLE 1 R¹—Y TARGETDYES MOLECULE R² R³ R⁴ R⁵—Y² R⁶ pK_(a) Me — Me Me Me Me Me 3.5 Et — EtEt Et Et Et 4.5 Me — Me Me Me HOCH₂CH₂ HOCH₂CH₂ 2.4 HOCH₂CH₂ — HOCH₂CH₂HOCH₂CH₂ HOCH₂CH₂ HOCH₂CH₂ HOCH₂CH₂ 2.3 Me — Me Me Me NO₂PhCH₂CH₂ Et 2.7NH₂C₆H₄CH₂CH₂ — Et Me Me Et Et 3.9 NH₂PhCH₂CH₂ — Et Et Et Et Et 4.4Me(OCH₂CH₂)₂ Me(OCH₂CH₂)₂ Me(OCH₂CH₂)₂ Me(OCH₂CH₂)₂ Me(OCH₂CH₂)₂Me(OCH₂CH₂)₂ 2.3 NH₂C₆H₄CH₂CH₂ Et Me(OCH₂CH₂)₂ Me(OCH₂CH₂)₂ Me(OCH₂CH₂)₂Me(OCH₂CH₂)₂ 2.4 Me(OCH₂CH₂)₂ Me(OCH₂CH₂)₂ Me(OCH₂CH₂)₂ Me(OCH₂CH₂)₂NH₂C₆H₄CH₂CH₂ Et 2.7 NH₂C₆H₄CH₂CH₂ Et Me(OCH₂CH₂)₂ Me(OCH₂CH₂)₂ Et Et4.3 COMPLEXES NHCSNHC₆H₄CH₂CH₂ Dextran Et Me Me Et Et 3.9NHCSNHC₆H₄CH₂CH₂ transferin Et Me Me Et Et 2.0 Me dextran Me Me MeNHCSNC₆H₄CH₂CH₂ Et 2.9 NHCSNHC₆H₄CH₂CH₂ transferin Et Me Me Et Et 2.0NH₂C₆H₄CH₂CH₂ BSA Et Me(OCH₂CH₂)₂ Me(OCH₂CH₂)₂ Et Et 2.2

The present invention is now illustrated by the following examples.

EXAMPLES

A variety of different dyes according to the present invention wereprepared as follows:

Example 1 Preparation of Starting Materials

Starting materials for the preparation of dyes according to the presentinvention and having the formulae (V) and (VI) and (VII) were preparedas shown in Scheme 1 and Scheme 2, respectively, below. The remainingstarting materials were prepared by standard chemical sytheses.

1a) N-(3-hydroxy-phenyl)-2-(4-nitrophenyl)-acetamide

Pyridine (14 g, 0.18 Mol) and 4-nitrophenylacetic acid (32 g, 0.18 Mol)were stirred in toluene (100 ml) whilst trimethylacetylchloride (21 g,0.18 Mol) was added. The toluene was evaporated to yield4-nitrophenylacetic-trimethylacetic anhydride. 3-Aminophenol (20 g, 0.18Mol) was stirred in DMF (100 ml) and treated with the mixed anhydrideand pyridine (14 g) in dimethylformamide (DMF) dropwise. The solutionwas diluted with water and the product isolated by filtration and dried(19 g, 39%).

1b) N-2(4-nitrophenyl)ethyl-N-(3-hydroxyphenyl)-acetamide

10 g (0.4 mol) of the nitrophenylacetanilide prepared in 1a) was placedin a flask and borane in tetrahydrofuran (THF) (1 M, 250 ml) was addedcarefully. The resulting solution was heated under reflux and water (12ml) in THF (100 ml) was added dropwise over 1 hour. A further 50 ml ofwater was added and the mixture was heated for 15 mins. The THF wasevaporated under reduced pressure and methanol (100 ml) was added todissolve the oil. Thin layer chromatography (tlc)(silica H, 20% methanolin chloroform) gave a R_(f) much higher than the starting acetanilide.

Sodium bicarbonate (64 g, 0.6 Mol) was then added with stirring,followed by acetic anhydride (20 ml, 0.2 Mol) dropwise. Tlc (silica H,30% ethylacetate in toluene) showed almost single spot R_(f) of 0.3. Thesolution was stirred for 2 days, after which the tlc showed that achange to a lower spot R_(f) of 0.2 had occurred. The suspension wasdiluted with water (50 ml) and the product recovered by filtration anddried (9.1 g). The product was then washed twice with 50 ml ether toremove impurities (6.7 g, 56%).

1c) N-ethyl-N-4-nitrophenylethyl-3-hydroxyaniline (V)

6 g of the N-2(4-nitrophenyl)ethyl-N-(3-hydroxyphenyl)acetamide preparedin lb) was treated with borane in THF as in 1b). After removal of theTHF, water was added and the product was recovered by filtration, washedwith aqueous methanol and dried to give a product having the formula (V)(5.7 g, 80%).

1d) Benzyl-3-nitrophenyl-ether

3-Nitrophenol (13.9 g) in DMF (30 ml) was treated with sodium carbonate(11 g) and benzybromide (17 g) under reflux. When the yellow colour ofthe reaction mixture had faded, 30% more carbonate and benzyl bromidewas added under reflux. When only a slight pale yellow remained,analysis by tlc confirmed the reaction to be complete. The productmixture was cooled; diluted with water (200 ml); extracted intoluene:petroleum (60-80) (1:1, 200 ml); washed with water (200 ml);dried with MgSO₄; and evaporated. The product was crystallised frommethanol (20 g, 87%)

Toluenesulphonyl chloride was added to 30% excess ofmethoxyethoxyethanol in pyridine and stirred overnight. 2 volumes ofwater was added, and the product recovered by filtration and dried.

1e) (3-Benzyloxy-phenyl)-bis-[2(2-methoxy-ethoxy)-ethyl]-amine

Benzyl-3-aminophenyl ether (2 g) and 2-(2-methoxy-ethoxy)-ethylp-toluenesulphonate (5.6 g) were heated under reflux in acetonitrile (5ml) and diisopropylethylamine (4 ml) overnight.

The reaction mixture was cooled and extracted into toluene, and thetoluene was then washed with saturated sodium bicarbonate, dried andevaporated. Flash chromatography (gradient of toluene to 75% ethylacetate in toluene over silica 60 (100 ml)) yielded some of the monoalkyl (0.25 g) and the required dialkyl amine (1.8 g, 44%).

1f) 3-{Bis-[2-(2-methoxy-ethoxy)-ethyl]-amino}-phenol (VI)

Benzyl-3-aminophenylether (1.8 g) was treated with 10% Pt/C (300 mg) inethanol (30 ml) with acetic acid (5 ml). Hydrogen uptake was complete in1 hour, and tlc (silica 60, 10% methanol in chloroform) showed a singlespot having a lower R_(f) than the starting benzylether (silica 60). Thecatalyst was removed by filtration, the solvent evaporated and theproduct dried in vacuo (1.4 g)

Example 2 Condensation to Form a Mixture of Dyes

Compound (V) (1 g, 3.4 mMol), compound (VI) (2 g, 6.4 mMol),5-nitrosalicylate methyl ester (0.5 g, 2.5 mMol) and silica gel 60 (4 g)were mixed and heated at 180° C. for 1 hour. The black powder obtainedwas cooled, more silica (10 g) was added and the mixture washed on afilter with ethyl acetate. The residue was extracted with aceticacid:chloroform:methanol (1:5:5), and the dark purple solutionevaporated to dryness (1.6 g). Analysis by tlc(dichloromethane:acetone:acetic acid, 60:30:10) showed three main purplebands, all slightly fluorescent when dipped in aqueous acetic acid, andall more fluorescent in dilute HCl. Column chromatography was performedon silica 60 (200 ml) eluting with 10% acetic acid in chloroform with apolarity gradient rising to 10% acetic acid in chloroform:acetone (1:2).After an initial brown material and a small purple band (including dyeA) had been eluted, fractions were collected. Those bands showing singlecomponents in the dichloromethane:acetone:acetic acid tlc wereevaporated to dryness to give highest R_(f) (dyes B1 and B2, M+1 875.5,45 mg), middle R_(f) (dyes C1 and C2, M+1 903.5, 80 mg) and lowest R_(f)(dye D, M+1 930.5, 30 mg) fractions.

Example 3 Reduction and Separation of Dyes C1 and C2 Having Low pK_(a)'sand Conversion to Isothiocyanates

10 mg of the purified material of intermediate R_(f) obtained in 2)above (i.e. a mixture of dyes C1 and C2) was dissolved in methanol (2ml) and acetic acid (200 μl), treated with stannous chloride (100 mg)and stirred overnight. Tlc in dichloromethane (DCM):acetone:acetic acid(60:30:10) showed two spots with reduced R_(f), both of which werestrongly fluorescent when dipped in HCl. The reaction mixture was thendiluted with HCl (0.1 M) and extracted with chloroform to give thehigher R_(f) material, i.e. the Cl amine shown below.

The reaction was repeated using 30 mg of a mixture of dyes C1 and C2from 2) above, with extraction from dilute HCl and washing to give theCl amine (8 mg), followed by neutralisation with aqueous sodium acetatesolution and extraction with chloroform and with methanol to give the C2amine (15 mg).

For bonding to a target molecule, such as a protein, the amino group ofthe dye is converted to an isothiocyanate group by reaction with excessthiocarbonyldiimidazole in the minimum of chloroform. The isothiocyanateis precipitated by the addition of diethyl ether and the productisolated by centrifugation or filtration.

Example 4 Exchange Reaction to Increase pK_(a), Followed by Reductionand Conversion to Isothiocyanate

Two reactions were performed as follows:

4a) A mixture of dinitro dyes B1 and B2 (30 mg) was treated withdiisopropylamine formate salt in DCM (1 M, 1 ml) and diethylaminophenol(140 mg, 1 mM).

4b) A mixture of mononitro dyes C1 and C2 (15 mg) was treated similarly.

The reactions were heated, with care to remove the DCM, and maintainedat 150° C. for 1 hour. Each reaction gave rise to two main new productsby tlc (silica H, DCM:acetone:acetic acid, 60:30:10), as shown below,with one product being common to both reactions and having an R_(f)intermediate the starting material bands. This material was isolated bydissolving in chloroform, absorbing onto silica gel 60 (ca. 1 g) andwashing with chloroform. The purple dye products were eluted withchloroform:acetone:acetic acid (45:45:20) and evaporated to dryness. Theresidues were applied to two tlc plates (silica H) and run inDCM:actone:acetic acid (60:40:10). The common band was removed andeluted with chloroform:methanol (1:1) and dried to give the exchangeproduct from B2 and C1 (ca. 5 mg), which had a pK_(a) of 4.2.

The reduction and conversion to the isothiocyanate was performed asabove.

Example 5 Labelling of Bovine Serum Albumin (BSA)

A dye having the formula (VII), below, has a fluorescence spectrum withan excitation maximum at 560 nm and an emission maximum at 585 nm, and afluorescence titration as shown in FIG. 1.

The dye was dissolved in DMSO (10 mg/ml) and 10 μl of the resultingsolution was added to a solution of defatted BSA (2 mg) in sodiumcarbonate buffer (50 μl, 100 mM, pH 8.5). The reaction was incubated at35° C. for two hours. The solution was neutralised by the addition ofacetic acid (1 M, 10 μl) and applied to a Sephadex G25 column (total bedvolume 2 ml) and, equilibrated with triethylamine acetate buffer (25 mM,pH 6). The labelled protein was eluted in the exclusion volume (ca. 1ml) and was collected and lyophilised.

Example 6 Labelling of Aminodextran

A dye having the formula (VIII), below, was synthesised in a mannersimilar to that described in 2) above, but starting with commerciallyavailable 3-dimethylaminophenol in place of thedi-methoxyethyoxyethylaminophenol (VI). This synthesis resulted in anisothiocyanate derivative with similar reporter properties but lackingsufficient solubility in aqueous media at neutral and higher pH, therebyprecluding its use for protein labelling.

0.5 mg of compound (VIII) was dissolved in DMSO (10 μl) and added toT-10 aminodextran (supplied by Molecular Probes, M_(r)=10 kDa, 5 mg)dissolved in DMSO (50 μl) in a glass centrifuge tube. The solution waskept at room temperature for 1.5 hours and the labelling reaction wasthen halted with the addition of acidified ethanol (100 μl HCl, 12 M, in10 ml ethanol). The precipitated amino dextran was recovered bycentrifugation (ca. 1000 G: 5 min) and the coloured supernatant solutionwas discarded. The precipitated pellet was then redissolved in DMSO (ca.100 μl) and acidified ethanol was added to precipitate and recover theaminodextran. The procedure was repeated three times until thesupernatant was colourless. The washed, labelled aminodextran was driedunder high vacuum (4.5 mg) then dissolved in a minimal volume ofdistilled H₂O (<100 μl).

A sample of this solution was then applied to a 2 ml Sephadex G25 columnthat had been pre-washed with buffer solution (20 mMtriethylamino-acetate in distilled water adjusted to pH 6.0 containingpolyethylene glycol 400 (2% v/v)). A single coloured band passed throughthe column in the exclusion volume (ca. 1.0 ml). A small sample of thedried, labelled aminodextran was analysed by thin layer chromatographyand no free dye was detected.

Example 7 Fluorescence-Labelled Aminodextran is Endocytosed andTrafficked to Lysosomes (Internal Acidic)

This experiment was performed using NRK cells (a kidney cell line) thathad been transfected with an over-expressing construct of a recombinantgene encoding a fusion protein between GFP (green fluorescent protein)and mucolipin. This construct ensures that the green fluorescence willbe localised together with the mucolipin in the membrane of thelysosomes. The recombinant cells were incubated with thefluorescence-labelled aminodextran, produced in 6) above, at ca. 1 mg/mlfor 1 hour and the medium was then replaced. At various time intervalsthereafter the cells were inspected by confocal fluorescence microscopyat the wavelengths appropriate for detecting both GFP (green) and dye(VIII) of the present invention (red).

FIG. 2 shows high magnification confocal microscopic images of part of asingle cell, showing lysosomes, taken after overnight incubation withthe fluororescence-labelled aminodextran.

The green fluorescence clearly shows GFP in the membrane of thevesicles. The red fluorescence shows fluorescence-labelled aminodextranin an acidic environment (required for fluorescence of the dye VIII).The red fluorescence (luminal) appears only within lysosomes outlined bygreen fluorescence (membrane-bound). This result is accentuated in boththe sum and difference images. Inspection of cells incubated withfluorescence-labelled aminodextran at earlier times showed little redfluorescence. However, all of the red fluorescence at early times wascentral to areas outlined with green fluorescence, as found at latertimes. This result also demonstrates the slow rate of traffic ofaminodextran into the lysosomes. In addition, the presence of someGFP-labelled lysosomes not showing red fluorescence indicates that somelysosomes are not reached by aminodextran even after 20 hours ofincubation.

Example 8 Preparation of2-[3,6-bis-dimethylaminoxanth-9-ylium]-5-diethylaminophenol

Di-[3-dimethylaminophenyl]ether (12 mg, 0.05 mM) and4-diethylaminosalicylaldehyde (9 mg, 0.05 mM) heated at 60° C. in HCl(100 μl, 2 M) for 1 day. Tlc (silica in chloroform:acetone:acetic acid60:40:5) showed virtually all starting material gone, a small quantityof purple spot at R_(f) 0.3 and a UV (254 nm) absorbing spot at R_(f)approximately 0.6, which became bright purple on UV irradiation; thiswas 2-[3,6-bis-dimethylaminoxanthen-9-yl]-5-diethylamino-pheno].

Two dimensional tlc in same solvent but with UV irradiation betweenelutions showed efficient conversion to the title compound. The reactionwas diluted into ethanol and irradiated with UV at approximately 350 nm.The solution turned darker red and tlc showed complete conversion of thecolourless band into the lower R_(f) purple band. The solution wasneutralised with potassium carbonate (colour change red fluorescent topurple) and extracted into chloroform. The chloroform extract waspurified by chromatography on two 20×20 silica G tlc plates run in 20%methanol, 5% acetic acid in chloroform. Extraction of the purple bandand evaporation gave the title compound (approximately 5 mg, 23%).

Example 9 9a) Preparation of3-diethylamino-3′-dimethylamino-2,2′-dihydroxydiphenylmethanol

4-Diethylaminosalicylaldehyde (193 mg, 1 mmole) and3-dimethylaminophenol (170 mg, 1.25 mmole) was dissolved in EtOH (10 ml)to which was added HCl (aq) (0.5 ml). The reaction was stirred at roomtemperature for 18 h. The solvent was removed in vacuo and the residuebasified. The product was extracted with CHCl₃₁ washed with H₂O, theorganic layers were combined, dried and the solvent removed in vacuo toyield, the title compound, a light blue solid (264 mg, 80%).

9b) Preparation of 3-diethylamino-6-dimethylamino-9-hydroxyxanthene

The acyclic methanol (IX) prepared in Example 9a (250 mg, 0.76 mmole)was dissolved in H₂O (5 ml) to which was added two drops of H₃PO₄ (70%).The solution was heated under reflux for 2 h. The reaction was cooledand an excess of solid Ba₂CO₃ was added while stirring. The mixture wasfiltered and the solvent removed in vacuo to afford the hydroxy pyroninbase (X) as a red solid (198.5 mg, 84%).

The pyronin base (X) may be converted to the corresponding ketone (XI,below) using methodology described by Ehrlich et al, Chem. Ber.(1913)46:1941.

9c) Preparation of2-[3,6-bis-dimethylaminoxanth-9-ylium]-5-dimethylaminophenol

Commercial pyronin Y having the formula (XI) above (dye content usuallyapproximately 50%) was extracted with hot ethanol, the solid impuritiesremoved by filtration and the solvent evaporated.

The purified material (56 mg, 0.2 mM) dissolved in water was treatedwith potassium carbonate solution (0.2 ml, approximately 4 M) and 5%Pt/C catalyst. The resulting suspension was stirred with EtOAc (5 ml)and treated with hydrogen peroxide (200 μl). When gas evolution finishedthe EtOAc layer was separated, and dried through a phase-separationfiltratration system and the solvent removed in vacuo to form (XI). Theresidue was dissolved in DCM and treated with dimethylaminophenol (28mg, 0.2 mM) and the solvent removed by heating to 80° C. After 4 hourstlc (silica, chloroform:acetone:acetic acid, 60:40:5) showed completeconversion to the dye (XII). The dye was separated from the excessaminophenol by chromatography (silica, 20% methanol in chloroform) togive the title compound (20 mg, 70%).

Example 10 One Pot preparation of2-[3-diethylamino-6-dimethylamino-xanth-9-ylium]-5-dimethylaminophenol

4-Diethylaminosalicylaldehyde (19 mg, 0.1 mmole), 3-dimethylaminophenol(17 mg, 0.13 mmole) were dissolved in H₂O (5 ml) to which was added 3drops of 70% H₃PO₄. The solution was stirred at 100° C. for 5 h. Tlc(silica, chloroform:acetone:acetic acid 60:40:5) showed loss of the twostarting amines and appearance of a lower R_(f) UV-absorbing compoundidentical to 3-diethylamino-6dimethylamino-9-hydroxyxanthene.3-Dimethylaminophenol (21 mg, 0.15 mmole) in 2 M aqueous HCl (1 ml) wasadded and the reaction was stirred for a further 2 h. Tlc showedappearance of a spot with further reduced R_(f). The solution wasexposed to UV light to form the title compound derivative that ran onthe same tlc at a much lower R_(f), very close to that of, and with thesame pH sensitivity as the closely related2-[3,6-bisdimethylaminoxanth-9-ylium]-5-dimethylaminophenol.

1. A fluorescent dye comprising a xanthene-derived fluorophore havingthe formula (I)

wherein R1, R2, R3, R4, R5 and R6 are independent selected from H,alkyl, alkoxy, alcohol, ether, alkenyl, alkenoxy, aryl, alkaryl, aralkyland amido, except that R1, R4 and/or R5 is not H when bonded to Y, Y1and/or Y2, respectively; X is either 0″ or S″; and at least one of Y,Y1, Y2, Y3, Y4 and Y5 is a group for covalently bonding the dye,optionally through the use of a coupling agent, to a target molecule,and is otherwise H.
 2. A dye according to claim 1, wherein X is 0′.
 3. Adye according to claim 1, wherein the target molecule is selected from apeptide, a polypeptide, a protein, a saccharide, a polysaccharide or anantibody.
 4. A dye according to claim 1, wherein the NR5R6 substituentis para to the xanthene moiety.
 5. A dye according to claim 1, which iswater soluble.
 6. A dye according to claim 5, wherein one or more of R1,R2, R3 or R4 is independently selected from alcohol, alkoxy and ethergroups.
 7. A dye according to claim 1, wherein Y, Y1 and/or Y2 isselected from —NCS; —NCO; —(CO)R wherein R is a leaving group H, OH oralkyl; —SH; and —S2H.
 8. A dye according to claim 1, wherein at leastone of Y and Y1 is a group for covalently bonding the dye to a targetmolecule, and ac d h of Y2, Y3, Y4 and Y5 is H.
 9. A dye according toclaim 1, wherein R1, R2, R3, R4, R5 and R6 are independently selectedfrom alkyl groups and X is 0″.
 10. A dye according to claim 9, whereinat least one of Y and Y1 is a group for covalently bonding the dye to atarget molecule, and each of Y2, Y3, Y4 and Y5 is H.
 11. A method formaking a dye as defined in claim 1, comprising reacting ameta-aminophenol with a carboxylate compound capable of undergoingalpha-cleavage.
 12. A method according to claim 11, wherein ameta-aminophenol is reacted with the beta-ketocarboxylatediethylmalonate.
 13. A method according to claim 11, wherein ameta-aminophenol is reacted with 5-nitrosalicylic acid methyl ester. 14.A method for making a dye having the formula (I)

wherein R1, R2, R3, R4, R5 and R6 are independently selected from H,alkyl, alkoxy, alcohol, ether, alkenyl, alkenoxy, aryl, alkaryl, aralkyland amido, except that R1, R4 and/or R5 is not H when bonded to Y, Y1and/or Y2, respectively; X is either 0″ or S″; and at least one of Y,Y1, Y2, Y3, and Y4 and Y5 is a group for covalently bonding the dye,optionally through the use of a coupling agent, to a target molecule,and is otherwise H: comprising reacting a compound having the formula(II)

with a compound of formula (CO)R7, wherein R7 is leaving group, to forman intermediate having the formula (III)

and converting the intermediate to a dye having the formula (I).
 15. Themethod of claim 14, further comprising reacting a compound having theformula (II) with a benzaldehyde, benzoic acid, activated benzoic acidor benzoate, wherein when reaction is with a benzaldehyde the resultingproduct is oxidised to form a dye having the formula (I).
 16. A methodfor making a dye having the formula (I)

wherein R1, R2, R3, R4, R5 and R6 are independently selected from H,alkyl, alkoxy, alcohol, ether, alkenyl, alkenoxy, aryl, alkaryl, aralkyland amido, except that R1, R4 and/or R5 is not H when bonded to Y, Y1and/or Y2, respectively; X is either 0″ or S″; and at least one of Y,Y1, Y2, Y3, Y4 and Y5 is a group for covalently bonding the dye,optionally through the use of a coupling agent, to a target molecule,and is otherwise H; comprising reacting together a para-aminosubstituted salicylaldehyde and a meta-aminophenol to form a compoundhaving the formula (IIa), wherein R8 is H or an alkyl group; cyclisingthe compound of formula (Ha) to form a compound having the formula(IIb); reacting the compound having the formula (IIb) with ameta-aminophenol to form a compound having the formula (IIc), andoxidising the compound having the formula (IIc) to form a dye having theformula (I)₂ wherein the compounds IIa, IIb, and IIc have the followingstructures:


17. A method for making a dye having the formula (I):

wherein R1, R2, R3, R4, R5 and R6 are independently selected from H,alkyl, alkoxy, alcohol, ether, alkenyl, alkenoxy, aryl, alkaryl, aralkyland amido, except that R1, R4 and/or R5 is not H when bonded to Y, Y1and/or Y2, respectively; X is either 0″ or S″; and at least one of Y,Y1, Y2, Y3, Y4 and Y5 is a group for covalently bonding the dye,optionally through the use of a coupling agent, to a target molecule,and is otherwise H; comprising reacting together a para-aminosubstituted salicylaldehyde and a meta-aminophenol to form a compoundhaving the formula (IIa):

cyclising the compound having the formula (IIa) to form a compoundhaving the formula (IIb)

converting the compound having the formula (Iib) to a compound havingthe formula (III)

and converting the compound having the formula (III) to a dye having theformula (I).
 18. The method of claim 16 further comprising reactingtogether a para-amino substituted salicylaldehyde and a meta-aminophenolto form a compound having the formula (IIa), and cyclising the compoundof formula (IIa) to form a compound having the formula (IIb).
 19. Amethod of making a dye having the formula (I):

wherein R1, R2, R3, R4, R5 and R6 are independently selected from H,alkyl, alkoxy, alcohol, ether, alkenyl, alkenoxy, aryl, alkaryl, aralkyland amido except that R1, R4 and/or R5 is not H when bonded to Y, Y1and/or Y2, respectively; X is either 0″ or S″; and at least one of Y,Y1, Y2, Y3, Y4 and Y5 is a group for covalently bonding the dye,optionally through the use of a coupling agent, to a target molecule,and is otherwise H; comprising reacting together a meta-aminophenol and,a compound having the formula

wherein X is a halogen, a B(OR9)2 group in which R9 is H or an alkylgroup or another compound group, to form a compound having the formula(II)

and converting the compound having the formula (II) to a dye having theformula (I) by: a) reaction with a salicylic ester; b) reaction with asalicylaldehyde, and subsequent oxidation; or c) converting to acompound having the formula (III):

and then converting this to a dye having the formula (I).
 20. A methodof making a dye having the formula (I):

wherein R1, R2, R3, R4, R5 and R6 are independently selected from H,alkyl, alkoxy, alcohol, ether, alkenyl, alkenoxy, aryl, alkaryl, aralkyland amido, except that R1, R4 and/or R5 is not H when bonded to Y, Y1and/or Y2, respectively; X is either 0″ or S″; and at least one of Y,Y1, Y2, Y3, Y4 and Y5 is a group for covalently bonding the dye,optionally through the use of a coupling agent, to a target molecule,and is otherwise H; comprising functionalizing a compound having theformula (IV)

wherein R1, R2, R3, R4, R5 and R6 are independently selected from H,alkyl, alkoxy, alcohol, alkenyl, alkenoxy, aryl, alkaryl, aralkyl, amidoand ether; X is O″ or S″; and one or more of the aryl rings optionallyincludes and additional substituent, so as to incorporate at least oneY, Y1, Y2, Y3, Y4 and Y5 as a group for covalently bonding the dye,optionally through a coupling agent, to a target molecule.
 21. Afluorescent complex comprising a dye as defined in claim 1, which isbonded through Y, Y1, Y2, Y3, Y4 or Y5 to a target molecule, optionallythrough a moiety derived from a coupling agent.
 22. A complex accordingto claim 21, wherein the target molecule is selected from a peptide, apolypeptide, a protein, a saccharide, a polysaccharide or an antibody.23. A complex according to claim 22, wherein, the fluorescent complex isbound to the target molecule through a linkage selected from —NH(CS)NH—;—NH(CO)—; —NHalkyl; —S(CS)NH—; —S(CO)—; —SS.
 24. A living cell or cellcompartment comprising the complex of claim
 21. 25. A method fordetermining the pH of a living cell or cell compartment comprising thesteps of mixing a dye having the formula (I);

wherein R1, R2, R3, R4, R5 and R6 are independently selected from H,alkyl, alkoxy, alcohol, ether, alkenyl, alkenoxy, aryl, alkaryl, aralkyland amido, except that R1, R4 and/or R5 is not H when bonded to Y, Y1and/or Y2, respectively; X is either 0″ or S″; and at least one of Y,Y1, Y2, Y3, Y4 and Y5 is a group for covalently bonding the dye,optionally through the use of a coupling agent, to a target molecule,and is H; or a fluorescent complex comprising the compound of formula(I) which is bound through Y, Y1, Y2, Y3, Y4 or Y5 to a target molecule,optionally through a moiety derived from a coupling agent, with a samplecomprising a living cell or cell compartment; allowing time for the dyeor complex to enter the cell or cell compartment; irradiating the cellor cell compartment; measuring the intensity of fluorescence of the cellor cell compartment; and determining the pH of the cell compartment. 26.A method according to claim 25, further comprising a second dye orcomplex having a different fluorescence response.
 27. A method foranalysing the kinetics of migration into a cell or cell compartment of adye having the formula (I);

wherein R1, R2, R3, R4, R5 and R6 are independently selected from H,alkyl, alkoxy, alcohol, ether, alkenyl, alkenoxy, aryl, alkaryl, aralkyland amido, except that R1, R4 and/or R5 is not H when bonded to Y, Y1and/or Y2, respectively; X is either 0″ or S″; and at least one of Y,Y1, Y2, Y3, Y4 and Y5 is a group for covalently bonding the dye,optionally through the use of a coupling agent, to a target molecule,and is otherwise H; or a fluorescent complex comprising the compound offormula (I) which is bound through Y, Y1, Y2, Y3, Y4 or Y5 to a targetmolecule, optionally through a moiety derived from a coupling agent, themethod comprising mixing said dye or complex with a sample comprising aliving cell or cell compartment; irradiating the cell or cellcompartment for a time interval; monitoring the intensity offluorescence of the dye or complex over the time interval; anddetermining the time for migration of the dye or complex into the cellor cell compartment.
 28. A method according to claim 27, furthercomprising a plurality of different dyes or complexes, each of which hasa different pKa; monitoring the intensity of fluorescence of each dye orcomplex over the time interval; and determining the time for migrationof each dye or complex into each cell or cell compartment.
 29. A methodaccording to claim 28, wherein the plurality of different dyes arebonded to a target molecule, wherein each dye has a differentfluorescent response.
 30. A pH testing kit comprising at least one dyehaving the formula (I):

wherein R1, R2, R3, R4, R5 and R6 are independently selected from H,alkyl, alkoxy, alcohol, ether, alkenyl, alkenoxy, aryl, alkaryl, andamido, except that R1, R4 and/or R5 is not H when bonded to Y, Y1 and/orY2, respectively; X is either 0″ or S″; and at least one of Y, Y1, Y2,Y3, Y4 and Y5 is a group for covalently bonding the dye, optionallythrough the use of a coupling agent, to a target molecule, and isotherwise H; or a fluorescent complex comprising the compound of formula(I) which is bound through Y, Y1, Y2, Y3, Y4 or Y5 to a target molecule,optionally through a moiety derived from a coupling agent and at leastone set of calibration data.
 31. A fluorescent dye having the formula(IV)

wherein R1, R2, R3, R4, R5 and R6 are independently selected from H,alkyl, alkoxy, alcohol, alkenyl, alkenoxy, aryl, alkaryl, aralkyl, amidoand ether; X is 0′ or S″; and one or more of the aryl rings optionallyincludes an additional substituent, with the proviso that when X is 0″not all of R1, R2, R3, R4, R5 and R6 are the same alkyl group unless anelectron withdrawing group is present in the aryl ring having the —NR5R6substituent, eg. para to the group X.
 32. A fluorescent dye according toclaim 31, with the proviso that when X is 0′ not all of R1, R2, R3, R4,R5 and R6 are alkyl.
 33. A method of making a fluorescent dye having theformula (IV)

wherein R1, R2, R3, R4, R5 and R6 are independently selected from H,alkyl, alkoxy, alcohol, alkenyl, alkenoxy, aryl, alkaryl, aralkyl, amidoand ether; X is O″ or S″; and one or more of the aryl rings, optionallyincludes an additional substituent, the method comprising reactingtogether a para-amino substituted salicyladehyde and a meta-aminophenolto form a compound having the formula (IId) wherein R8 is H or alkylgroup, cyclising the compound of formula (IId) to form a compound havingthe formula (IIe); reacting the compound of formula (IIe) with ameta-aminophenol to form a compound having the formula (IIf); andoxidising the compound having the formula (IIf) to form a dye having theformula (IV); wherein, IId, IIe, and IIf having the followingstructures:


34. A method according to claim 33, wherein in the dye having theformula (IV) at least one of R1, R2, R3 and R4 is different from theothers.
 35. A method according to claim 33, wherein R1, R2, R3, R4, R5and R6 are independently selected from alkyl groups.
 36. A method formaking a compound having the formula (IIe):

wherein R1, R2, R3, and R4 are independently selected from H, alkyl,alkoxy, alcohol, alkenyl, alkenoxy, aryl, alkaryl, amido and ether; X isO″ or S″; and one or more of the aryl rings optionally includes anadditional substituent; R8 is H or an alkyl group; comprising reactingtogether para-amino substituted salicyladehyde and a meta-aminophenol toform a compound having the formula (IId):

and cyclising the compound having the formula (IId) to form a compoundhaving the formula (IIe).